We have screened for homozygous deletions that occur during tumor progression, using Representational Difference Analysis (RDA), a PCR-based genomic subtractive technique that effectively isolates DNA fragments that are absent in tumor cells, compared with matched normal tissues. The small size of homozygous deletions, compared with the large hemizygous losses that define "LOH," have made RDA an effective approach to identify novel tumor suppressor genes. The recent sequencing of the human (and eventually mouse) genomes has greatly enhanced the power of this approach, allowing rapid identification of genes located within deleted loci. We have used a syngeneic mouse tumor model to avoid the polymorphic microdeletions that complicate RDA analysis using human samples. The p53/NF2-double heterozygous null mouse tumor model reproducibly generates highly metastatic tumors from which multiple cell lines have been established. We have validated the application of RDA in this mouse model by correctly identifying the p16INK4a locus, which is deleted in a subset of these tumor cell lines, as well as a new homozygous deletion. We have identified a novel gene, DOS, which spans this deletion and encodes a homolog of Dock180, implicated in Rac1 signaling. We propose to characterize DOS and search for additional homozygous deletions in our panel of cell lines. In Specific Aim 1, we will address the general relevance of DOS in cancer by searching for point mutations in mouse and human cancer cell lines and in primary human tumor specimens. In Specific Aim 2, we will make use of our DOS-null cell line to test the functional properties of wild-type and mutant DOS proteins. Proteins known to interact with Dock180 will be tested for association with DOS, and novel protein interactors will be sought. In Specific Aim 3, we will expand our RDA screen by studying an additional 30 tumor/normal pairs by RDA, providing a panel of deleted probes for further genomic mapping and gene identification. Taken together, this project will aim to uncover new genes whose homozygous inactivation contributes to tumor progression. The relevance of these new candidate tumor suppressors in human cancer will be addressed by mutational and expression studies; these new candidate tumor suppressors in human cancer will be addressed by mutational and expression studies; genetic and RNAi studies in C. elegans and Drosophila will be used to provide initial insight into their functional properties.